The RNA exosome shapes the expression of key protein-coding genes.

The ribonucleolytic exosome complex is central for nuclear RNA degradation, primarily targeting non-coding RNAs. Still, the nuclear exosome could have protein-coding (pc) gene-specific regulatory activities. By depleting an exosome core component, or components of exosome adaptor complexes, we identify ∼2900 transcription start sites (TSSs) from within pc genes that produce exosome-sensitive transcripts. At least 1000 of these overlap with annotated mRNA TSSs and a considerable portion of their transcripts share the annotated mRNA 3' end. We identify two types of pc-genes, both employing a single, annotated TSS across cells, but the first type primarily produces full-length, exosome-sensitive transcripts, whereas the second primarily produces prematurely terminated transcripts. Genes within the former type often belong to immediate early response transcription factors, while genes within the latter are likely transcribed as a consequence of their proximity to upstream TSSs on the opposite strand. Conversely, when genes have multiple active TSSs, alternative TSSs that produce exosome-sensitive transcripts typically do not contribute substantially to overall gene expression, and most such transcripts are prematurely terminated. Our results display a complex landscape of sense transcription within pc-genes and imply a direct role for nuclear RNA turnover in the regulation of a subset of pc-genes.

TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.

Telomerase-mediated telomere elongation provides cell populations with the ability to proliferate indefinitely. Telomerase is capable of recognizing and extending the shortest telomeres in cells; nevertheless, how this mechanism is executed remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced upon telomere shortening is polyadenylated and largely devoid of telomeric repeats, and furthermore, telomerase physically interacts with this polyadenylated TERRA in vivo We also show that experimentally enhanced transcription of a manipulated telomere promotes its association with telomerase and concomitant elongation. Our data represent the first direct evidence that TERRA stimulates telomerase recruitment and activity at chromosome ends in an organism with human-like telomeres.

Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency.

Heterogeneous expression of the transcription factor NANOG has been linked to the existence of various functional states in pluripotent stem cells. This heterogeneity seems to arise from fluctuations of Nanog expression in individual cells, but a thorough characterization of these fluctuations and their impact on the pluripotent state is still lacking. Here, we have used a novel fluorescent reporter to investigate the temporal dynamics of NANOG expression in mouse embryonic stem cells (mESCs), and to dissect the lineage potential of mESCs at different NANOG states. Our results show that stochastic NANOG fluctuations are widespread in mESCs, with essentially all expressing cells showing fluctuations in NANOG levels, even when cultured in ground-state conditions (2i media). We further show that fluctuations have similar kinetics when mESCs are cultured in standard conditions (serum plus leukemia inhibitory factor) or ground-state conditions, implying that NANOG fluctuations are inherent to the pluripotent state. We have then compared the developmental potential of low-NANOG and high-NANOG mESCs, grown in different conditions, and confirm that mESCs are more susceptible to enter differentiation at the low-NANOG state. Further analysis by gene expression profiling reveals that low-NANOG cells have marked expression of lineage-affiliated genes, with variable profiles according to the signalling environment. By contrast, high-NANOG cells show a more stable expression profile in different environments, with minimal expression of lineage markers. Altogether, our data support a model in which stochastic NANOG fluctuations provide opportunities for mESCs to explore multiple lineage options, modulating their probability to change functional state.

Lin28a is dormant, functional, and dispensable during mouse oocyte-to-embryo transition.

The oocyte-to-embryo transition (OET) denotes transformation of a highly differentiated oocyte into totipotent blastomeres of the early mammalian embryo. OET depends exclusively on maternal RNAs and proteins accumulated during oocyte growth, which implies importance of post-transcriptional control of gene expression. OET includes replacement of abundant maternal microRNAs (miRNAs), enriched also in differentiated cells and exemplified by the Let-7 family, with embryonic miRNAs common in pluripotent stem cells (the miR-290 family in the mouse). Lin28a and its homolog Lin28b encode RNA-binding proteins, which interfere with Let-7 maturation and facilitate reprogramming of induced pluripotent stem cells. Both Lin28a and Lin28b transcripts are abundant in mouse oocytes. To test the role of maternal expression of Lin28a and Lin28b during oocyte-to-zygote reprogramming, we generated mice with oocyte-specific knockdown of both genes by using transgenic RNA interference. Lin28a and Lin28b are dispensable during oocyte growth because their knockdown has no effect on Let-7a levels in fully grown germinal vesicle (GV)-intact oocytes. Furthermore, transgenic females were fertile and produced healthy offspring, and their overall breeding performance was comparable to that of wild-type mice. At the same time, 2-cell embryos derived from transgenic females showed up-regulation of mature Let-7, suggesting that maternally provided LIN28A and LIN28B function during zygotic genome activation. Consistent with this conclusion is increased translation of Lin28a transcripts upon resumption of meiosis. Our data imply dual repression of Let-7 during OET in the mouse model, the selective suppression of Let-7 biogenesis by Lin28 homologs superimposed on previously reported global suppression of miRNA activity.

[Colorectal cancer and canonical Wnt signalling pathway]. / Kolorektální karcinom a kanonická Wnt signalizace.

Colorectal cancer is among the most frequent malignancies in the economically developed part of the world including the Czech Republic. From the molecular biological point of view, the development of colorectal cancer is in most cases linked to the pathologically activated canonical Wnt signalling pathway. The aim of this review is to present this intercellular signalling pathway, its role in the colorectal carcinogenesis and possible therapeutic implications according to our current knowledge.

Does a Hospital Palliative Care Team Have the Potential to Reduce the Cost of a Terminal Hospitalization? A Retrospective Case-Control Study in a Czech Tertiary University Hospital.

Background: More than 50% of patients worldwide die in hospitals and end-of-life care is costly. We aimed to explore whether support from the palliative team can influence end-of-life costs. Methods: This was a descriptive retrospective case-control study conducted at a Czech tertiary hospital. We explored the difference in daily hospital costs between patients who died with and without the support of the hospital palliative care team from January 2019 to April 2020. Big data from registries of routine visits were used for case-control matching. As secondary outcomes, we compared the groups over the duration of the terminal hospitalization, intensive care unit (ICU) days, intravenous antibiotics, magnetic resonance imaging/computed tomography scans, oncological treatment in the last month of life, and documentation of the dying phase. Standard descriptive statistics were used to describe the data, and differences between the case and control groups were tested using Fisher's exact test for categorical variables and the Mann-Whitney U test for numerical data. Results: In total, 213 dyads were identified. The average daily costs were three times lower in the palliative group (4392.4 CZK per day = 171.3 EUR) than in the nonpalliative group (13992.8 CZK per day = 545.8 EUR), and the difference was probably associated with the shorter time spent in the ICU (16% vs. 33% of hospital days). Conclusions: We showed that the integration of the palliative care team in the dying phase can be cost saving. These data could support the implementation of hospital palliative care in developing countries.

RNA-DNA Hybrids Support Recombination-Based Telomere Maintenance in Fission Yeast.

A subset of cancers rely on telomerase-independent mechanisms to maintain their chromosome ends. The predominant "alternative lengthening of telomeres" pathway appears dependent on homology-directed repair (HDR) to maintain telomeric DNA. However, the molecular changes needed for cells to productively engage in telomeric HDR are poorly understood. To gain new insights into this transition, we monitored the state of telomeres during serial culture of fission yeast (Schizosaccharomyces pombe) lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). In this context, degradation of TERRA associated with the telomere in the form of R-loops drives a severe growth crisis, ultimately leading to a novel type of survivor with linear chromosomes and altered cytological telomere characteristics, including the loss of the shelterin component Rap1 (but not the TRF1/TRF2 ortholog, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective in this context, preventing the growth crisis that is otherwise triggered by degradation of telomeric R-loops in survivors with linear chromosomes. These findings suggest that upregulation of telomere-engaged TERRA, or altered recruitment of shelterin components, can support telomerase-independent telomere maintenance.

Inflammation regulates 11ß-hydroxysteroid dehydrogenase type 1 differentially in specific compartments of the gut mucosal immune system.

The bioavailability of glucocorticoids is modulated by enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11HSD1), which catalyzes the conversion of inactive 11-oxo-glucocorticoids to active 11-hydroxy-glucocorticoids cortisol and corticosterone and is regulated by pro-inflammatory cytokines. Our aim was to assess the effect of colitis on the expression of 11HSD1 in specific microanatomical compartments of the mucosal immune system. Using qRT-PCR we quantified the expression of 11HSD1 and cytokines in the colon, mesenteric lymph nodes (MLN) and spleen of mice with colitis. Microsamples of the MLN cortex, paracortex and medulla, colonic crypt epithelium (CCE), lamina propria and isolated intestinal lymphoid follicles (ILF) were harvested by laser microdissection, whereas splenic and MLN lymphocytes by flow cytometry. Colitis increased 11HSD1 in the CCE, ILF, and MLN cortex but not in the lamina propria and the MLN paracortex and medulla. Expression of IL-4, IL-21 and TNFα was increased in both the cortex of MLN and ILF, whereas IL-1ß and IL-10 were only increased in the follicles. No positive effect was observed in the case of IFNγ and TGFß. 11HSD1 was positively correlated with TNFα and less strongly with IL-21, IL-1ß, and IL-4. Colitis also upregulated the 11HSD1 expression of T cells in the spleen and MLN. The study demonstrates the stimulatory effect of inflammation on local glucocorticoid metabolism only in particular compartments of the mucosal immune system. The correlation between cytokines and 11HSD1 in the ILF and MLN cortex indicates that pro-inflammatory cytokines may amplify glucocorticoid signals in inductive compartments of the mucosal immune system.

Expression of 11ß-hydroxysteroid dehydrogenase type 2 is deregulated in colon carcinoma.

Although the effects of glucocorticoids on proliferation, differentiation and apoptosis are well known, and steroid hormones have been identified to play a role in pathogenesis and the development of various cancers, limited data are available regarding the relationship between the local metabolism of glucocorticoids and colorectal adenocarcinoma (CRC) formation. Glucocorticoid metabolism is determined by 11ß-hydroxysteroid dehydrogenases type 1 and 2 (11HSD1, 11HSD2), which increase the local concentration of cortisol due to the reduction of cortisone, or decrease this concentration due to the oxidation of cortisol. The objective of this study was to evaluate the extent of 11HSD1 and 11HSD2 mRNA in pre-malignant colorectal polyps and in CRC. The specimens were retrieved from patients by endoscopic or surgical resection and the expression of 11HSD1 and 11HSD2 was measured by real-time PCR. The polyps were of the following histological types: hyperplastic polyps and adenomas with low- or high-grade dysplasia. The neoplastic tissue of CRC obtained during tumor surgery was also studied. It was found that 11HSD2 was not only downregulated in CRC but already in the early stages of neoplastic transformation (adenoma with low-grade dysplasia). In contrast, the level of 11HSD1 was significantly increased in CRC but not in pre-malignant polyps. The results demonstrate that the downregulation of 11HSD2 gene expression is a typical feature of the development of colorectal polypous lesions and their transformation into CRC.

Deep sequencing reveals complex spurious transcription from transiently transfected plasmids.

Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls.